1. Field of the Invention
The present invention relates to a method for determining nucleotide sequences of single nucleic acid molecules by single molecule detection.
2. Description of the Background
The Sanger method (Proc. Natl. Acad. Sci. USA, 74:5463, 1977) is routinely used as a method for determining a nucleotide sequence of DNA. This method is also referred to as the dideoxy chain termination method, entailing the steps of annealing a primer to the 5′ end of a DNA sample, synthesizing a complementary chain in the presence of DNA polymerase and four deoxynucleoside triphosphates (dNTPs where N denotes A, C, T and G) and each of 2′, 3′-dideoxynucleoside triphosphates (ddNTPs where N denotes A, C, T or G), stopping the elongation reaction at a position where ddNTP is incorporated, and performing gel electrophoresis for the obtained reaction product, thereby determining a nucleotide sequence of the DNA sample. The DNA fragments formed at this time are generally labeled with a radioactive label, which enables the identification of the position of the fragments.
Furthermore, since radioactive labeling requires a special facility, other methods for determining nucleotide sequences which employ fluorescent labels in place of radioactive labels to detect fluorescence by irradiation of a laser beam, have also been developed (e.g. Japanese Patent No. 2901004, and JP-B-7-43347).
However, the use of the Sanger method remains problematic as, for example it requires production of a large number of copies of the DNA to be sequenced by previously incorporating the DNA into a vector, e.g. M13, and that the number of detectable bases is limited to a maximum 1,000 bp per lane of electrophoresis. Hence, if the nucleotide sequence of a DNA could be determined by directly identifying the bases from the 5′ end of the sequence one by one, and an isolated single DNA molecule could be used for direct sequencing without requiring a number of replicated molecules of DNA, efficiency of sequencing could, thereby, be significantly improved.